Use of toll-like receptor and agonist for treating cancer

ABSTRACT

The present invention is directed to methods and agents used for treating cancer or infectious diseases by providing toll-like receptors such as toll-like receptor 5 (TLR-5) in combination with providing a toll-like receptor agonists such as flagellin resulting in a cis and intrans effect that recruits cells involved in both the innate (cis effect) and adaptive (trans effect) immune response to specifically kill cancer cells and cells infected with a pathogen via the NF-κB apoptosis pathway.

FIELD OF THE INVENTION

This invention relates to methods of treating cancer and infectiousdiseases.

BACKGROUND OF THE INVENTION

Toll-like receptors are responsible for the recognition of most commonpatterns of bacterial and viral pathogens. Their activation results inrecruitment of innate and subsequently adaptive immune response.Receptor cells of the immune system to the site of presence of antigensis the key step in effective immune response. That is why immunizationinvolves the use of different types of adjuvants. Although the majorityof tumors express tumor-specific antigens, they are using a number ofmechanisms allowing then to escape immune recognition. It was recentlydemonstrated in mouse models that activation of TLR5 by its ligand andagonist, bacterial flagellin, results in the induction of antitumoreffect against those tumors that express functional TLR5. This opens ageneral opportunity for considering TLR5 agonists for cancerimmunotherapy. There are two major obstacles on the way to reduction ofthis idea to practice. First, is the rare incidence of tumors expressingfunctional TLR5 limiting applicability of this approach to only a smallsubset of tumors. Second, systemic administration of TLR5 agonist leadsto activation of TRL5 signaling in all cells that have functionalreceptor making response unfocused and not tumor-specific. Accordingly,there is a need in the art for a mechanism or method for autocrineactivation of TLR receptor signaling in infected or tumor cells withminimal systemic effect thus enabling to attract innate immune responsespecifically to the infected cell or tumor.

SUMMARY OF THE INVENTION

The present invention may be directed to a vector comprising a first andsecond nucleic acid, wherein the first nucleic acid encodes a toll-likereceptor and the second nucleic acid encodes a toll-like receptoragonist. The first nucleic acid may encode for a secreted form of atoll-like receptor. The second nucleic acid may be a secreted form offlagellin. The toll-like receptor agonist may be flagellin. The vectormay be a mammalian expression vector. The vector may be expressed froman adenovirus, a lentivirus or a liposome. The secreted form offlagellin may be CBLB502S. The toll-like receptor may be TLR-5.

The present invention may be directed to a method of treating cancer ina mammal comprising administering to a mammal in need thereof a agentcomprising the vector comprising a first and second nucleic acid,wherein the first nucleic acid encodes a toll-like receptor and thesecond nucleic acid encodes a toll-like receptor agonist. The cancer maybe a tumor. The tumor may be derived from the group consisting ofprostate, breast, colon, esophagus, stomach, lung, pancreatic, renal,thyroid, ovaries, throat, or the cervix. The tumor may be derived fromthe group consisting of sarcomas, melenomas, leukemias, and lymphomas.The agent may be administered in trans or outside from the tumor of themammal. The agent may be administered directly into a tumor of themammal. The agent may be administered in combination with animmunostimulant. The immunostimulant may be selected from the groupconsisting of growth hormone, prolactin and vitamin D. The growthhormone may be somatotrophin. The agent may be administered incombination with a cytokine. The cytokine may be a stem cell factor.

The present invention is also directed to a method for treating aninfection in a mammal comprising administering to a mammal in needthereof the agent of agent comprising the vector comprising a first andsecond nucleic acid, wherein the first nucleic acid encodes a toll-likereceptor and the second nucleic acid encodes a toll-like receptoragonist. The cancer may be a tumor. The infection may be derived fromthe group consisting of viruses, bacteria, protozoan parasites andfungi.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C depicts schematic maps of adenoviral vectors expressing TLR5,CBLB502S and their combination (TLR5+CBLB502S).

FIG. 2 depicts the results of the ratio of tumor volume in mice over anumber of days in tumor cells (A549) transduced with a control vector(without TLR5) or vector expressing TLR5 wherein the mice are treatedthree days with either CBLB502 or PBS.

FIG. 3 depicts suppression of tumor growth by injection of adenoviruscomprising vector coexpressing CBLB502S and Toll-like receptor whereinthe adenovirus is injected into syngeneic mice CT26 colon carcinomatumor and studying the in-cis and in-trans effects of the adenoviralvector constructs.

FIG. 4 shows the domain structure of bacterial flagellin. The Cabackbone trace, hydrophobic core distribution and structural informationof F41. Four distinct hydrophobic cores that define domains D1, D2a, D2band D3. All the hydrophobic side-chain atoms are displayed with the Cabackbone. Side-chain atoms are color coded: Ala, yellow; Leu, Ile orVal, orange; Phe and Tyr, purple (carbon atoms) and red (oxygen atoms).c, Position and region of various structural features in the amino-acidsequence of flagellin. Shown are, from top to bottom: the F41 fragmentin blue; three b-folium folds in brown; the secondary structuredistribution with a-helix in yellow, b-structure in green, and b-turn inpurple; tic mark at every 50th residue in blue; domains D0, D1, D2 andD3; the axial subunit contact region within the proto-element in cyan;the well-conserved amino-acid sequence in red and variable region inviolet; point mutations in F41 that produce the elements of differentsupercoils. Letters at the bottom indicate the morphology of mutantelements: L (D107E, R124A, R124S, G426A), L-type straight; R (A449V),R-type straight; C (D313Y, A414V, A427V, N433D), curly33.

FIG. 5 shows a schematic of Salmonella flagellin domains, its fragments,and its interaction with TLR5. Dark bars denote regions of the flagellingene used to construct fragments comprising A, B, C, A′ and B′.

FIG. 6 depicts flagellin derivatives. The domain structure andapproximate boundaries (amino acid coordinates) of selected flagellinderivatives (listed on the right). FliC flagellin of Salmonella dublinis encoded within 505 amino acids (aa).

FIG. 7 shows the nucleotide and amino acid sequence for the followingflagellin variants: AA′ (SEQ ID NO: 7-8), AB′ (SEQ ID NO: 9-10), BA′(SEQ ID NO: 11-12), BB′ (SEQ ID NO: 13-14), CA′ (SEQ ID NO: 15-16), CB′(SEQ ID NO: 17-18), A (SEQ ID NO: 19-20), B (SEQ ID NO: 21-22), C (SEQID NO: 23-24), GST-A′ (SEQ ID NO: 25-26), GST-B′ (SEQ ID NO: 27-28),AA′n1-170 (SEQ ID NO: 29-30), AA′n1-163 (SEQ ID NO: 33-34), AA′n54-170(SEQ ID NO: 31-32), AA′n54-163 (SEQ ID NO: 335-36), AB′n1-170 (SEQ IDNO: 37-38), AB′n1-163 (SEQ ID NO: 39-40), AA′n1-129 (SEQ ID NO: 41-42),AA′n54-129 (SEQ ID NO: 43-44), AB′n1-129 (SEQ ID NO: 45-46), AB′n54-129(SEQ ID NO: 47-48), AA′n1-100 (SEQ ID NO: 49-50), AB′n1-100 (SEQ ID NO:51-52), AA′n1-70 (SEQ ID NO: 53-54) and AB′n1-70 (SEQ ID NO: 55-56). ThepRSETb leader sequence is shown in Italic (leader includes Met, which isalso amino acid 1 of FliC). The N terminal constant domain isunderlined. The amino acid linker sequence is in Bold. The C terminalconstant domain is underlined. GST, if present, is highlighted.

FIG. 8 shows a comparison of amino acid sequences of the conserved amino(FIG. 8A) and carboxy (FIG. 8B) terminus from 21 species of bacteria.The 13 conserved amino acids important for TLR5 activity are shown withshading. The amino acid sequences are identified by their accessionnumbers from TrEMBL (first letter=Q) or Swiss-Prot (first letter=P).

FIG. 9 shows the nucleic acid and amino acid sequence for the humanToll-like receptor 5 protein.

DETAILED DESCRIPTION

The inventors have made the surprising discovery that the provision of atoll-like receptor, such as toll-like receptor 5 (TLR-5), in combinationwith a toll-like receptor agonist, such as flagellin, results in a cisand in-trans effect that recruits cells involved in both the innate (ciseffect) and adaptive (trans effect) immune response to specifically killcancer cells and cells infected with a pathogen via the NF-κB apoptosispathway. While not being bound by theory, the idea implemented in thisinvention was to (i) overcome the dependence of TLR-mediatedimmunization strategies on pre-existing TLR expression in a tumor bytransducing the tumor with a construct driving expression of TLR; and(ii) to direct the immune response to the tumor by creating local poolof TLR agonist. For example, drug formulations comprising TLRsimultaneously induce expression and activate TLR, thereby exposingtumor cells to the host immune system imitating the situation of massivebacterial penetration through the intestinal wall.

By providing a TLR such as TLR5, and a TLR agonist such as flagellin, tointeract and activate both the innate and adaptive immune system, themethod can be used to treat tumors derived from the prostate, breast,colon, esophagus, stomach, lung, pancreatic, renal, thyroid, ovaries,throat, or the cervix cancer as well as treating sarcomas, melenomas,leukemias, and lymphomas. Applications of this method are not limited tocancer treatments, as this method can also be used to treat infectionsderived from viruses, bacteria, protozoan parasites and fungi.

Variations of providing the TLR and TLR agonist may include vectors,co-expressing the TLR receptor and a secretable form of flagellin thatactivates TLR activity in the same compromised mammalian cell. Themethod of the present invention may also include vector constructs thatexpress the TLR receptor in a mammalian cell and the TLR agonist beingadministered in trans to the cell. For example, an adenoviral vector mayrequire modification of flagellin to reach its effective synthesis andsecretion by mammalian cells.

1. Definitions.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitlycontemplated.

“Administer” may mean a single dose or multiple doses of an agent oragent.

“Analog” may mean, in the context of a peptide or polypeptide, a peptideor polypeptide comprising one or more non-standard amino acids or otherstructural variations from the conventional set of amino acids.

“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, orfragments, or derivatives thereof, including Fab, F(ab′)2, Fd, andsingle chain antibodies, diabodies, bispecific antibodies, bifunctionalantibodies and derivatives thereof. The antibody may be a monoclonalantibody, polyclonal antibody, affinity purified antibody, or mixturesthereof which exhibits sufficient binding specificity to a desiredepitope or a sequence derived therefrom. The antibody may also be achimeric antibody. The antibody may be derivatized by the attachment ofone or more chemical, peptide, or polypeptide moieties known in the art.The antibody may be conjugated with a chemical moiety.

A “derivative” may mean a peptide or polypeptide different other than inprimary structure (amino acids and amino acid analogs). Derivatives maydiffer by being glycosylated, one form of post-translationalmodification. For example, peptides or polypeptides may exhibitglycosylation patterns due to expression in heterologous systems. If atleast one biological activity is retained, then these peptides orpolypeptides are derivatives according to the invention. Otherderivatives may include fusion peptides or fusion polypeptides having acovalently modified N- or C-terminus, PEGylated peptides orpolypeptides, peptides or polypeptides associated with lipid moieties,alkylated peptides or polypeptides, peptides or polypeptides linked viaan amino acid side-chain functional group to other peptides,polypeptides or chemicals, and additional modifications as would beunderstood in the art.

A “fragment” may mean a portion of a reference peptide or polypeptide.

A “homolog” may mean a peptide or polypeptide sharing a commonevolutionary ancestor.

A “leader sequence” may be a nucleic acid encoding any peptide sequencethat is linked and translated with a peptide or polypeptide of interestto allow the peptide or polypeptide of interest be properly routedthrough a eukaryotic cell's endoplasmic reticulum and Golgi complexesfor the purposed of extracellular secretion from the cell's membrane.The leader peptide sequence may be derived from alkaline phosphatase.The leader sequence may have a DNA sequence comprisingatgctgctgctgctgctgctgctgggcctgaggctacagctct ccctgggc.

A “liposome” may mean a tiny bubble (vesicle) made out of the samematerial as a cell membrane. A liposome be filled with drugs and used todeliver drugs for cancer and other diseases. A liposome may be filledwith a vector. A liposome membrane may be made of phospholipids, whichare molecules that have a head group and a tail group. The head of theliposome may be attracted to water, and the tail, which is made of along hydrocarbon chain, is repelled by water. The tails may be repelledby water, and line up to form a surface away from the water. The lipidsin the plasma membrane may be chiefly phospholipids likephosphatidylethanolamine and phosphatidylcholine. Liposomes may becomposed of naturally-derived phospholipids with mixed lipid chains(like egg phosphatidylethanolamine), or of pure surfactant componentslike DOPE (dioleoylphosphatidylethanolamine).

A “peptide” or “polypeptide” may mean a linked sequence of amino acidsand may be natural, synthetic, or a modification or combination ofnatural and synthetic.

“Substantially identical” may mean that a first and second amino acidsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%,or 99% over a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100 amino acids .

“Treating,” “treatment,” or “to treat” each may mean to alleviate,suppress, repress, eliminate, prevent or slow the appearance ofsymptoms, clinical signs, or underlying pathology of a condition ordisorder on a temporary or permanent basis. Preventing a condition ordisorder involves administering a agent of the present invention to asubject prior to onset of the disease. Suppressing a condition ordisorder involves administering a agent of the present invention to asubject after induction of the condition or disorder but before itsclinical appearance. Repressing the condition or disorder involvesadministering a agent of the present invention to a subject afterclinical appearance of the disease.

A “variant” may mean means a peptide or polypeptide that differs inamino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. Representative examples of “biological activity” include theability to bind to a toll-like receptor and to be bound by a specificantibody. Variant may also mean a protein with an amino acid sequencethat is substantially identical to a referenced protein with an aminoacid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporatedfully herein by reference. Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art.Substitutions may be performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hyrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.

A “vector” may mean a nucleic acid sequence containing an origin ofreplication. A vector may be a plasmid, a yeast or a mammalianartificial chromosome. A vector may be a RNA or DNA vector. A vector maybe either a self-replicating extrachromosomal vector or a vector whichintegrates into a host genome.

2. Toll-Like Receptor

Provided herein is a toll-like receptor (TLR), which may be a type ofpattern recognition receptor (PRR). The TLR may recognize molecules thatare conserved molecular products derived from pathogens that includeGram-positive, Gram-negative bacteria, fungi, and viruses, but aredistinguishable from host molecules, collectively referred to aspathogen-associated molecular patterns (PAMPs). The TLR may alsorecognize endogenous molecules released from injured or dying cells,collectively referred to as damage-associated molecular pattern (DAMPs).A PAMP or DAMP may be a TLR agonist as further described below. The TLRmay be a fragment, variant, analog, homolog or derivative that recruitsadapter molecules within the cytoplasm of cells in order to propagate asignal. The TLR may be from a human or other mammalian species such asrhesus monkey, mouse, or rat. The TLR may be at least 30-99% identicalto a TLR that recruits adapter molecules within the cytoplasm of cellsin order to propagate a signal.

The TLR may be one of the between ten and fifteen types of TLR that areestimated to exist in most mammalian species. The TLR may be one of the13 TLR (named simply TLR1 to TLR13) that have been identified in humansand mice together, or may be an equivalent form that has been found inother mammalian species. The TLR may be one of the 11 members(TLR1-TLR11) that have been identified in humans.

The TLR may be expressed by different types of immune cells, and may belocated on the cell surface or in the cell cytoplasm. The TLR may beexpressed on cancer cells. The TLR may be expressed by normal epithelialcells in the digestive system, normal keratinocytes in the skin,alveolar and bronchial epithelial cells, and epithelial cells of thefemale reproductive tract. These cells lining an organ may be the firstline of defense against invasion of micoorganisms, and TLRs expressed inepithelial cells may have a crucial role in the regulation ofproliferation and apoptosis.

The TLR-expressing cancer cell may be selected from the following table:

TABLE 1 TLR expression in Human Cancer cells TYPE OF CANCER TLR Gastriccancer TLR2, TLR4, TLR5, TLR9 Colorectal cancer TLR2, TLR3, TLR4, TLR5,TLR9 Ovarian cancer TLR2, TLR3, TLR4, TLR5 Cervical cancer TLR3, TLR4,TLR5, TLR9 Lung cancer TLR2, TLR3, TLR4, TLR9 Prostate cancer TLR4, TLR9Melanoma TLR2, TLR3, TLR4 Brain cancer TLR2, TLR4 Breast cancer TLR2,TLR3, TLR4, TLR9 Hepatocellular carcinoma TLR2, TLR3, TLR4, TLR6, TLR9Laryngeal cancer TLR2, TLR3, TLR4

The TLR expressed on cancer cells may upregulate the NF-κB cascade andproduce anti-apoptotic proteins that contribute to carcinogenesis andcancer cell proliferation.

Four adapter molecules of TLRs are known to be involved in signaling.These proteins are known as myeloid differentiation factor 88 (MyD88),Tirap (also called Mal), Trif, and Tram. The adapters activate othermolecules within the cell, including certain protein kinases (IRAK1,IRAK4, TBK1, and IKKi) that amplify the signal, and ultimately lead tothe induction or suppression of genes that orchestrate the inflammatoryresponse. TLR signaling pathways during pathogen recognition may induceimmune reactions via extracellular and intracellular pathways mediatedby MyD88, nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB), and mitogen-associated protein kinase (MAPK). In all, thousandsof genes are activated by TLR signaling, and collectively, the TLRconstitute one of the most pleiotropic, yet tightly regulated gatewaysfor gene modulation.

TLRs together with the Interleukin-1 receptors form a receptorsuperfamily, known as the “Interleukin-1 Receptor/Toll-Like ReceptorSuperfamily.” All members of this family have in common a so-called TIR(Toll-IL-1 receptor) domain. Three subgroups of TIR domains may exist.Proteins with subgroup I TIR domains are receptors for interleukins thatare produced by macrophages, monocytes and dendritic cells and all haveextracellular Immunoglobulin (Ig) domains. Proteins with subgroup II TIRdomains are classical TLRs, and bind directly or indirectly to moleculesof microbial origin. A third subgroup of proteins containing TIR domains(III) consists of adaptor proteins that are exclusively cytosolic andmediate signaling from proteins of subgroups 1 and 2. The TLR may be afragment, variant, analog, homolog or derivative that retains either asubgroup I TIR domain, subgroup II TIR domain, or subgroup III TIRdomain.

The TLR may function as a dimer. For example, although most TLRs appearto function as homodimers, TLR2 forms heterodimers with TLR1 or TLR6,each dimer having a different ligand specificity. The TLR may alsodepend on other co-receptors for full ligand sensitivity, such as in thecase of TLR4′s recognition of LPS, which requires MD-2. CD14 and LPSBinding Protein (LBP) are known to facilitate the presentation of LPS toMD-2.

a. TLR1

The TLR may be TLR1, which recognizes PAMPs with a specificity forgram-positive bacteria. TLR1 has also been designated as CD281.

b. TLR5

The TLR may be Toll-like receptor 5. The protein encoded by the TLR-5may play a fundamental role in pathogen recognition and activation ofinnate immunity. TLR-5 may recognize PAMPs that are expressed oninfectious agents, and mediate the production of cytokines necessary forthe development of effective immunity. TLR-5 may recognize bacterialflagellin, a principal component of bacterial flagella and a virulencefactor. The activation of the TLR may mobilize the nuclear factor NF-κBand stimulate tumor necrosis factor-alpha production.

3. Toll-like Receptor Agonist

Also provided herein is a TLR agonist. The TLR agonist may be a PAMP,which may be conserved molecular product derived from a pathogen. Thepathogen may be a Gram-positive bacterium, Gram-negative bacterium,fungus, or virus. The TLR agonist may be a damage-associated molecularpattern (DAMP) ligand, which may be an endogenous molecule released frominjured or dying cells. A DAMP or PAMP may initiate an immune responsethrough TLR signals and recruit adapter molecules within the cytoplasmof cells in order to propagate a signal. The TLR agonist may be anagonist for the TLR, which may be a ligand from the following in Table2:

TABLE 2 TLRs and Ligands TLR Ligand DAMP Ligand PAMP TLR1 Triacyllipoproteins TLR2 Heat Shock proteins Peptidoglycan HMGB1 (high mobilityLipoprotein group box 1—amphoterin) Lipoteichoic acid Zymosan TLR3 SelfdsRNA Viral dsRNA TLR4 Heat shock proteins Heat shock proteinsFibrinogen Lipopolysaccharides Heparan sulfate RSV fusion proteinFibronectin MMTV (Mouse mammary tumor virus) envelope proteinsHyaluronic acid Paclitaxel HMGB1 TLR5 flagellin TLR6 Lipoteichoic acidTriacyl lipoproteins zymosan TLR7/TLR8 Self ssRNA Viral ssRNA TLR9 SelfDNA Bacterial and viral DNA TLR10 TLR11 Profilin

The TLR agonist may be a fragment, variant, analog, homology orderivative of a PAMP or DAMP that binds a TLR and induces TLR-mediatedactivity, such as activation of NF-κB activity. The TLR agonsistfragment, variant, analog, homolog, or derivative may be at least 30-99%identical to amino acids of a TLR-agonist and induce TLR-mediatedactivity.

The TLR agonist may target a TLR such as TLR-5. The TLR agonist may bean agonist of TLR-5 and stimulate TLR-5 activity. The TLR agonist may bean anti-TLR5 antibody or other small molecule. The TLR agonist may beflagellin.

The flagellin may also be a flagellin or flagellin-related polypeptide.The flagellin may be from any source, including a variety ofGram-positive and Gram-negative bacterial species. The flagellin may bea flagellin polypeptide from any Gram-positive or Gram-negativebacterial species including, but not limited to, a flagellin polypeptidedisclosed in U.S. Pat. Pub. No. 2003/000044429, the contents of whichare fully incorporated herein by reference. For example, the flagellinmay have an amino acid sequence from a bacterial species depicted inFIG. 7 of U.S. Patent Publication No. 2003/0044429. The nucleotidesequences encoding the flagellin polypeptides listed in FIG. 7 of U.S.2003/0044429 are publicly available at sources including the NCBIGenbank database. The flagellin may also be a flagellin peptidecorresponding to an Accession number listed in the BLAST results shownin FIG. 25 of U.S. Patent Pub. 2003/000044429, or a variant thereof. Theflagellin may also be a flagellin polypeptide as disclosed in U.S.Patent Appl. Publication No. 2009/0011982, the contents of which arefully incorporated herein. The flagellin maybe anyone of a flagellinpolypeptide as disclosed in FIGS. 6 and 7 herein.

The flagellin may be a fragment, variant, analog, homology or derivativeof a flagellin that binds TLR5 and induces TLR5-mediated activity, suchas activation of NF-κB activity. A fragment, variant, analog, homolog,or derivative of flagellin may be at least 30-99% identical to aminoacids of a flagellin that binds TLR5 and induces TLR5-mediated activity.

The flagellin may be from a species of Salmonella, a representativeexample of which is S.dublin (encoded by GenBank Accession NumberM84972). The flagellin related-polypeptide may be a fragment, variant,analog, homolog, or derivative of M84972, or combination thereof, thatbinds to TLR5 and induces TLR5-mediated activity, such as activation ofNF-kB activity. A fragment, variant, analog, homolog, or derivative offlagellin may be obtained by rational-based design based on the domainstructure of Flagellin and the conserved structure recognized by TLR5.

The flagellin may comprise at least 10, 11, 12, or 13 of the 13conserved amino acids shown in FIG. 5 (positions 89, 90, 91, 95, 98,101, 115, 422, 423, 426, 431, 436 and 452). The flagellin may be atleast 30-99% identical to amino acids 1 174 and 418 505 of M84972. FIG.26 of U.S. Patent Appl Publication No. 2009/0011982, the contents ofwhich are fully incorporated herein, lists the percentage identity ofthe amino- and carboxy-terminus of flagellin with known TLR-5stimulating activity, as compared to M84972.

The flagellin may be the major component of bacterial flagellum. Theflagellin may be composed of three domains (FIG. 4). Domain 1 (D1) anddomain 2 (D2) may be discontinuous and may be formed when residues inthe amino terminus and carboxy terminus are juxtaposed by the formationof a hairpin structure. The amino and carboxy terminus comprising the D1and D2 domains may be most conserved, whereas the middle hypervariabledomain (D3) may be highly variable. Studies with a recombinant proteincontaining the amino D1 and D2 and carboxyl D1 and D2 separated by anEscherichia coli hinge (ND1-2/ECH/CD2) indicate that D1 and D2 may bebioactive when coupled to an ECH element. This chimera, but not thehinge alone, may induce IkBa degradation, NF-kB activation, and NO andIL-8 production in two intestinal epithelial cell lines. Thenon-conserved D3 domain may be on the surface of the flagellar filamentand may contain the major antigenic epitopes. The potent proinflammatoryactivity of flagellin may reside in the highly conserved N and C D1 andD2 regions (See FIG. 4).

The flagellin may induce NF-kB activity by binding to Toll-like receptor5 (TLR5). The TLR may recognize a conserved structure that is particularto the flagellin. The conserved structure may be composed of a largegroup of residues that are somewhat permissive to variation in aminoacid content. Smith et al., Nat Immunol. 4:1247-53 (2003), the contentsof which are incorporated herein by reference, have identified 13conserved amino acids in flagellin that are part of the conservedstructure recognized by TLR5. The 13 conserved amino acids of flagellinthat may be important for TLR5 activity are shown in FIG. 5.

Numerous deletional mutants of flagellin have been made that retain atleast some TLR5 stimulating activity. The flagellin may be such adeletional mutant, and may be a deletional mutant disclosed in theExamples herein. The flagellin may comprise a sequence translated fromGenBank Accession number D13689 missing amino acids 185-306 or 444-492,or from GenBank Accession number M84973 missing amino acids 179-415, ora variant thereof.

The flagellin may comprise transposon insertions and changes to thevariable D3 domain. The D3 domain may be substituted in part, or inwhole, with a hinge or linker polypeptide that allows the D1 and D2domains to properly fold such that the variant stimulates TLR5 activity.The variant hinge elements may be found in the E. coli MukB protein andmay have a sequence as set forth in SEQ ID NOS: 3 and 4, or a variantthereof.

The flagellin as described above may further comprise a leader sequence.The flagellin further comprising a leader sequence may be CBLB502S.

4. Agent

This invention also relates to an agent comprising a therapeuticallyeffective amount of a TLR and TLR agonist. The agent may deliver the TLRseparately from the TLR agonist. The agent may be a vector. The vectormay comprise a first nucleic acid encoding the TLR and a second nucleicacid comprising the TLR agonist. The vector may be capable oftransducing mammalian cells. The vector may be capable of bi-cistronicexpression of the TLR and/or TLR agonist using strong promoters. Thevector may comprise only a gene encoding the TLR, which may becontrolled by a strong promoter. The vector may be delivered into amammalian cell by a virus or liposome related vector system. The virusvector system may be an adenovirus or a cytomegalovirus.

The agent may be a liposome harboring the vector. The liposome maybecapable of transducing mammalian cells and delivering the vector forexpression.

The agent may be a drug formulation that simultaneously inducesexpression and activates the TLR, thereby exposing tumor or infectedcells to the host immune system imitating the situation of a massivepenetration through the intestinal wall. The agent may be a drugformulation that expresses the TLR in combination with the TLR agonist,and may be delivered systematically in solution for administration suchas intramuscularly. The agent may be a drug formulation that expressesthe TLR in combination with the TLR agonist, which may be expressed fromthe same vector, such as an adenoviral or cytomegalovirus vector system.The agent may be a drug formulation that expresses the TLR incombination with the TLR agonist expressed in the form of anano-particle, which may carry a functional agonist to the cell surfaceof a mammalian cell.

The agent may be a pharmaceutical agent comprising the drug formulationdescribed above, which may be produced using methods well known in theart. The agent may also comprise a coagent.

The vector may comprise a first nucleic acid encoding TLR5 and a secondnucleic acid comprising flagellin. The vector may be capable ofexpressing TLR5 and/or flagellin using a strong promoter. The expressionvector may further comprise a leader sequence cloned upstream of thegene encoding the TLR or TLR5 and/or flagellin. The expression vectormay be pCD515 based vector system. The expression vector may bepCD515-CMV-hTLR5-EF1-502 as described in FIG. 1A. The expression vectormay be pCD515-CMV-hTLR5 as described in FIG. 1B. The expression vectormay be pCD515-CMV-Sseap-502 as described in FIG. 1C.

The agent may be drug formulation that simultaneously induces expressionand activates a TLR thereby exposing tumor or infected cells to the hostimmune system imitating the situation of a massive penetration throughthe intestinal wall. The drug formulation may be in the form of a viralexpression system harboring the vector. The drug formulation may be anadenovirus expression functional human TLR5 in combination with:

the TLR agonist, delivered systematically in solution foradministration, such as intramuscularly;

the TLR agonist, expressed from the same adenoviral vector as the TLR;or

the TLR agonist, expressed in the form of nano-particles carryingfunctional TLR agonist, such as flagellin, which may be derived fromCBLB502, on their surface. The nano-particle may be on the basis of abacteriophage T7, or fully formed to retain its biological activity. Thenano-formulation may provide for dose-dependent, NF-κB-responsivereporter activation, and may result in cell internalization byendocytosis for effective immunization approach (Mobian AP-A).

a. Administration

Administration of the agents using the method described herein may beorally, parenterally, sublingually, transdermally, rectally,transmucosally, topically, via inhalation, via buccal administration, orcombinations thereof. Parenteral administration includes, but is notlimited to, intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intrathecal, and intraarticular. For veterinary use, theagent may be administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal. The agents may be administered to ahuman patient, cat, dog, large animal, or an avian.

The agent may be administered simultaneously or metronomically withother treatments. The term “simultaneous” or “simultaneously” as usedherein, means that the agent and other treatment be administered within48 hours, preferably 24 hours, more preferably 12 hours, yet morepreferably 6 hours, and most preferably 3 hours or less, of each other.The term “metronomically” as used herein means the administration of theagent at times different from the other treatment and at a certainfrequency relative to repeat administration.

The agent may be administered at any point prior to another treatmentincluding about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr,106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50hr, 48 hr,46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr,4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35mins., 30 mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins, 8 mins, 7mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins. The agentmay be administered at any point prior to a second treatment of theagent including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr,108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50hr,48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr,6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35mins., 30 mins., 25 mins., 20 mins., 15 mins., 10 mins., 9 mins., 8mins., 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins.

The agent may be administered at any point after another treatmentincluding about 1 min, 2 mins., 3 mins., 4 mins., 5 mins., 6 mins., 7mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins., 25 mins., 30mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1 hr, 2 hr, 3hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr,24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42 hr, 44hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 hr, 56 hr, 58 hr, 60 hr, 62 hr, 64hr, 66 hr, 68 hr, 70 hr, 72 hr, 74 hr, 76 hr, 78 hr, 80 hr, 82 hr, 84hr, 86 hr, 88 hr, 90 hr, 92 hr, 94 hr, 96 hr, 98 hr, 100 hr, 102 hr, 104hr, 106 hr, 108 hr, 110 hr, 112 hr, 114 hr, 116 hr, 118 hr, and 120 hr.The agent may be administered at any point prior after a secondtreatment of the agent including about 120 hr, 118 hr, 116 hr, 114 hr,112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54hr, 52 hr, 50hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr,32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45mins., 40 mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins., 10mins., 9 mins., 8 mins., 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2mins, and 1 mins.

b. Formulation

The method may comprise administering the agent. Agents provided hereinmay be in the form of tablets or lozenges formulated in a conventionalmanner. For example, tablets and capsules for oral administration maycontain conventional excipients may be binding agents, fillers,lubricants, disintegrants and wetting agents. Binding agents include,but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth,mucilage of starch and polyvinylpyrrolidone. Fillers may be lactose,sugar, microcrystalline cellulose, maizestarch, calcium phosphate, andsorbitol. Lubricants include, but are not limited to, magnesiumstearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants may be potato starch and sodium starch glycollate. Wettingagents may be sodium lauryl sulfate. Tablets may be coated according tomethods well known in the art.

Agents provided herein may also be liquid formulations such as aqueousor oily suspensions, solutions, emulsions, syrups, and elixirs. Theagents may also be formulated as a dry product for constitution withwater or other suitable vehicle before use. Such liquid preparations maycontain additives such as suspending agents, emulsifying agents,nonaqueous vehicles and preservatives. Suspending agent may be sorbitolsyrup, methyl cellulose, glucose/sugar syrup, gelatin,hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel,and hydrogenated edible fats. Emulsifying agents may be lecithin,sorbitan monooleate, and acacia. Nonaqueous vehicles may be edible oils,almond oil, fractionated coconut oil, oily esters, propylene glycol, andethyl alcohol. Preservatives may be methyl or propyl p-hydroxybenzoateand sorbic acid.

Agents provided herein may also be formulated as suppositories, whichmay contain suppository bases such as cocoa butter or glycerides. Agentsprovided herein may also be formulated for inhalation, which may be in aform such as a solution, suspension, or emulsion that may beadministered as a dry powder or in the form of an aerosol using apropellant, such as dichlorodifluoromethane or trichlorofluoromethane.Agents provided herein may also be formulated as transdermalformulations comprising aqueous or nonaqueous vehicles such as creams,ointments, lotions, pastes, medicated plaster, patch, or membrane.

Agents provided herein may also be formulated for parenteraladministration such as by injection, intratumor injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The agent may also be provided in apowder form for reconstitution with a suitable vehicle including, butnot limited to, sterile, pyrogen-free water.

Agents provided herein may also be formulated as a depot preparation,which may be administered by implantation or by intramuscular injection.The agents may be formulated with suitable polymeric or hydrophobicmaterials (as an emulsion in an acceptable oil, for example), ionexchange resins, or as sparingly soluble derivatives (as a sparinglysoluble salt, for example).

c. Dosage

The method may comprise administering a therapeutically effective amountof the agent to a patient in need thereof. The therapeutically effectiveamount required for use in therapy varies with the nature of thecondition being treated, the length of time desired to activate TLRactivity, and the age/condition of the patient. In general, however,doses employed for adult human treatment typically are in the range of0.001 mg/kg to about 200 mg/kg per day. The dose may be about 1 mg/kg toabout 100 mg/kg per day. The desired dose may be convenientlyadministered in a single dose, or as multiple doses administered atappropriate intervals, for example as two, three, four or more sub-dosesper day. Multiple doses may be desired, or required.

The dosage may be at any dosage such as about 0.1 mg/kg, 0.2 mg/kg, 0.3mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg,1 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 425 mg/kg, 450 mg/kg, 475 mg/kg,500 mg/kg, 525 mg/kg, 550 mg/kg, 575 mg/kg, 600 mg/kg, 625 mg/kg, 650mg/kg, 675 mg/kg, 700 mg/kg, 725 mg/kg, 750 mg/kg, 775 mg/kg, 800 mg/kg,825 mg/kg, 850 mg/kg, 875 mg/kg, 900 mg/kg, 925 mg/kg, 950 mg/kg, 975mg/kg or 1 mg/kg.

5. Method for Treating Cancer

Provided herein is a method for treating cancer by administering to amammal in need thereof the agent. The method provide immunotherapyagainst cancer by conversion of tumor cells into a TLRagonist-responsive state with targeted intratumor stimulation of TLR,thereby focusing an immune response on the tumor. The method may be beused to treat primary tumors prior to surgical removal in order toreduce the risk of metastasis development, as well as treat of othertumor nodules. The method may comprise intratumor injection. The methodmay have the step of injecting the agent into a primary tumor prior tosurgical removal to reduce the risk of metastasis development, as wellas treat other tumor nodules. The method may be used to treat any tumorthat is accessible for adenovirus intratumor injection.

A variety of cancers may be treated according to this invention,including carcinoma, bladder (including accelerated and metastaticbladder cancer), breast, colon (including colorectal cancer), kidney,liver, lung (including small and non-small cell lung cancer and lungadenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphaticsystem, rectum, larynx, pancreas (including exocrine pancreaticcarcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin(including squamous cell carcinoma); hematopoietic tumors of lymphoidlineage including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocyticlymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineageincluding acute and chronic myelogenous leukemias, myelodysplasticsyndrome, myeloid leukemia, and promyelocytic leukemia; tumors of thecentral and peripheral nervous system including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal originincluding fibrosarcoma, rhabdomyoscarcoma, and osteosarcoma; and othertumors including melanoma, xenoderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer, teratocarcinoma, and cancers of thegastrointestinal tract or the abdominopelvic cavity.

The method may be combined with other methods for treating cancer,including use of an immunostimulant, cytokine, or chemotherapeutic. Theimmunostimulant may be a growth hormone, prolactin or vitamin D.

6. Treatment of Infected Cells

Provided herein is a method for treating an infectious disease by thesimultaneous delivery of transduced cells by the agent. The method maybe used to treat a viral, bacterial, protozoan parasite or fungalinfection. The method may be used to treat any infectious disease byusing intracellular injection resulting in autocrine activation of TLRsignaling of infected cells with minimal systemic effect and therebyenabling to attract innate immune response specific to the infectedcells. The method may be combined with other therapies for treatingviral, bacterial, protozoan parasite or fungi infections.

The method may comprise administering the agent. The method may compriseadministration of a vaccine comprising the agent, and may be used incombination with any other vaccination, which may comprise a constructexpressing an antigen of choice.

EXAMPLE 1 Synthesis of Bi-cistronic Expression TLR5/flagellin Vector andTreatment of Tumor Cells

Vector constructs were created for expressing Toll-like receptor 5(TLR-5) and flagellin CBLB502. Vector pCD515 was used as a backbone forthese constructs. The cDNA sequence of human TLR-5 and the DNA encodingthe toll-like receptor agonist's CBLB502 were individually fused withleader peptide derived from alkaline phosphatase enabling routing of theexpressed protein through the endoplasmic reticulum (ER) and Golgitowards extracellular secretion.

The pCD515-CMV-hTLR5-EF1-502s vector construct expressed the secretedform of CBLB502 flagellin (CBLB502S) and the toll-like receptor 5 (TLR5)at the cell surfcace. This adenoviral vector required modification ofthe CBLB502 to reach its effective synthesis and secretion by mammaliancells. The adenovirus construct comprises the leader nucleic acidsequence (Atgctgctgctgctgctgctgctgggcctgaggctacagctctccctgggc) derivedfrom alkaline phosphatase and was cloned upstream of the truncatedSalmonella flagellin (fliC) gene (see Burdelya et al., Science320:226-230 (2008) to encode a secretable form of flagellin (i.e.,CBLB502S). An EF1 (elongation factor la) promoter was cloned upstream ofthis cassette encoding CBLB502S. The TLR5 gene was derived from humanand has the amino acid sequence as shown in FIG. 9. A CMV promoter wascloned upstream of the TLR5 gene. This construct co-expresses TLR5 andCBLB502S. This construct is shown in FIG. 1A.

The pCD515-CMV-hTLR5 expression vector was constructed to express theform of human TLR-5 (see FIG. 9). The adenovirus construct comprises astrong CMV promoter cloned upstream of the hTLR5 cassette. Thisconstruct is shown in FIG. 1B.

The pCD515-CMV-Sseap-502 expression vector was constructed to expressthe secreted flagellin CBLB502 and the toll-like. The adenovirusconstruct comprises a strong CMV promoter cloned upstream of the leadersequence SEAP 502 flagellin (fliC) gene. This construct is shown in FIG.1C. [Need cloning information]

EXAMPLE 2 Synthesis of Bi-cistronic Expression TLR5/flagellin Vector andTreatment of Tumor Cells

Two reporter mammalian cell lines, both expressing NF-kB-responsive GFPand differing in their TLR5 status, were transduced with vectorconstructs pCD515, pCD515-CMV-hTLR5-EF1-502s, pCD515-CMV-hTLR5-502,pCD515-CMV-hTLR5, and pCD515-CMV-Sseap-502 (see Table 3 below).

TABLE 3 Activity of adenoviral constructs as TLR5 signaling activatorsReport Reporter Line-293- Line-293- Treatment null TLR CBLB502 − + Ad5(control) (pCD515) − − Ad5 (TLR5) (pCD515-CMV-hTLR5) − − Ad5(TLR5) +CBLB502 + + (pCD515-CMV-hTLR5-EF1-502) Ad5 (CBLB502S) − +(pCD515-CMV-Sseap-502) Ad5 (TLR5) (pCD515-CMV- + + hTLR5) +Ad5(CBLB502S) (pCD515-CMV-hTLR5-EF1-502s) Ad5 (CBLB502S + TLR5) + +(pCD515-CMV-hTLR5-EF1-502s)

Vector co-expressing TLR5 and TLR5 agonist CBLB502S was sufficient toinduce expression of NF-kB reporter in 293-null cells that do notexpress any of known TLRs and which cannot be activated by TLR5 agonistalone. This experiment demonstrates that TLR5 and flagellin CBLB502S canwork in trans or in cis to activate TLR5 signaling.

EXAMPLE 3

To test antitumor effects of bi-cistronic adenovirus having(pCD515-CMV-hTLR5-EF1-502s), 10 ml of the adenoviral suspension(1012-1011 IU/ml) were injected into one of two s.c. growing syngeneictumors in Balb/c mice originating from CT26 mouse colon carcinoma cellswhen tumors reached 3-5 mm in diameter and tumor size was monitoreduntil control non-injected tumors reached size limit requiringtermination of the experiment. Control mice were injected (again, onetumor out of two per mouse) with adenoviral vector expressing redfluorescent protein (RFP). The results of a representative experimentare shown in FIG. 4. Almost complete lack of growth of tumors injectedwith (pCD515-CMV-hTLR5-EF1-502s) was accompanied with reduced growth ofthe uninjected tumor within the same animal as compared with the tumorsin control animals injected with RFP-expressing adenovirus. This resultindicates (i) powerful in-cis and (ii) visible in-trans effect ofpCD515-CMV-hTLR5-EF1-502s indicative of recruitment of both innate (ciseffect) and adaptive (trans effect) immune response. Neither of theother control viruses listed in Table 1 (i.e., ADS (control) and Ad5(TLR5)) injected alone had growth suppressive effects on tumors.

Thus, enforced ectopic expression of TLRS makes tumor cell types, whichoriginally were TLRS deficient, highly responsive to TLRS stimulationresulting in breaking tumor immuno-tolerance, powerful attraction ofinnate immune response that promotes effective development of adaptiveimmune response with subsequent general antitumor effect.

1. A vector comprising a first and second nucleic acid, wherein thefirst nucleic acid encodes a toll-like receptor and the second nucleicacid encodes a toll-like receptor agonist.
 2. The vector of claim 1,wherein the toll-like receptor agonist is flagellin.
 3. The vector ofclaim 1, wherein the vector is an expression vector.
 4. The vector ofclaim 3, wherein the vector is a mammalian expression vector.
 5. Thevector of claim 3, wherein the vector is expressed from an adenovirus, alentivirus or a liposome.
 6. The vector of claim 1, wherein the firstnucleic acid is a secreted form of a toll- like receptor.
 7. The vectorof claim 2 wherein the flagellin is a secreted form of flagellin.
 8. Thevector of claim 7, wherein the secreted form of flagellin comprises thethirteen conserved amino acids of flagellin that may be important forTLR5 activity are shown in FIG.
 5. 9. The vector of claim 1, wherein thetoll-like receptor is TLR-5.
 10. The vector of claim 1, wherein thefirst nucleic acid comprises a sequence as shown in FIG. 7 and thesecond nucleic acid comprises a sequence as shown in FIG.
 9. 11. Amethod of treating cancer in a mammal comprising administering to amammal in need thereof an agent comprising the vector of claim
 1. 12.The method of claim 11, wherein the cancer is a tumor.
 13. The method ofclaim 12, wherein the tumor is derived from the group consisting ofprostate, breast, colon, esophagus, stomach, lung, pancreatic, renal,thyroid, ovaries, throat, or the cervix.
 14. The method of claim 12,wherein the tumor is derived from the group consisting of sarcomas,melenomas, leukemias, and lymphomas.
 15. The method of claim 12, whereinthe agent is administered in trans from the tumor of the mammal.
 16. Themethod of claim 12, wherein the agent is administered directly into atumor of the mammal.
 17. The method of claim 11, wherein the agent isadministered in combination with an immunostimulant.
 18. The method ofclaim 11, wherein the immunostimulant is selected from the groupconsisting of growth hormone, prolactin and vitamin D.
 19. The method ofclaim 18, wherein the growth hormone is somatotrophin.
 20. The method ofclaim 11, wherein the agent is administered in combination with acytokine.
 21. The method of claim 20, wherein the cytokine is stem cellfactor.
 22. A method for treating an infection in a mammal comprisingadministering to a mammal in need thereof an agent comprising the vectorof claim
 1. 23. The method of claim 22, wherein the infection is by anorganism selected from the group consisting of a virus, a bacterium, aprotozoan parasites, and a fungus.